1. Field of the Invention
The invention pertains to materials and methods that may be used in investigating the mechanisms of apoptosis and, more particularly, nucleases that are implicated in apoptotic DNA degradation. The invention also further relates to materials and methods that may be useful for identifying therapeutic agents that target the apoptotic pathways.
2. Description of the Related Art
As used in the discussion below, references by author name and publication year are more particularly cited in the References section. Programmed cell death or apoptosis is needed for the development and tissue homeostasis of metazoans, for example, as discussed in Steller (1995); and Vaux and Korsmeyer (1999). Despite its significant role in many biological processes; apoptosis and its associated biochemical pathways are poorly understood. There is a need to improve understanding about apoptosis pathways for the development of useful pharmacological agents.
One step in apoptosis is the fragmentation of chromosomal DNA at internucleosomal regions. This fragmentation generates DNA ladders approximately 180 base pairs (bp) in length, as described by Wyllie (1980); and Zhang and Xu (2002). Several nucleases have been implicated in mediating this chromosome fragmentation processes, including DFF40/CAD, a 40 kD DNA fragmentation factor (DFF)/Caspase-Activated Deoxyribonuclease (CAD) (see Enari et al., (1998); Liu et al. (1997); and Liu et al. (1998)) and mitochondrial endonuclease G (Endo G) (see Li et al., 2001; and Parrish et al. (2001). Parrish et al. (2001) is incorporated by reference to the same extent as though fully replicated herein.
DFF40/CAD normally associates tightly with its cognate inhibitor, DFF45/ICAD, but is activated during apoptosis when it is released from DFF45/ICAD as a result of caspase cleavage of DFF45/ICAD. In contrast, Endo G is released from mitochondria and translocates to nuclei during apoptosis to induce DNA fragmentation in a manner that is independent of caspase and DFF40, as reported by Li et al. (2001). These different mechanisms show that multiple DNA degradation pathways exist. In addition, several other mammalian proteins, including apoptosis-inducing factor (AIF), DNaseII, Topoisomerase II, and cyclophilins have been implicated in mediating apoptotic DNA degradation, mostly based on in vitro studies, as described in Zhang and Xu (2002).
In the nematode C. elegans, at least two nucleases have been shown to mediate apoptotic DNA degradation: NUC-1, a worm type-II DNase discussed in Wu et al. (2000), and CPS-6, which is the C. elegans ortholog of Endo G described in Parrish et al. (2001). Loss-of-function mutations in either cps-6 or nuc-1 genes result in accumulation of TUNEL (for “‘TdT-mediated dUTP nick end labeling’”)-positive nuclei in mutant embryos. Thus, both CPS-6 and NUC-1 proteins appear to function to play a role in resolving 3′OH DNA breaks labeled by TUNEL, that are generated during apoptosis, as described in Parrish et al. (2001; and Wu et al. (2000). In addition, cell deaths in the cps-6 mutant are delayed, and sometimes blocked in sensitized genetic backgrounds, suggesting that the DNA degradation process is implicated in apoptosis, as reported by Parrish et al. (2001).
Unlike cps-6, nuc-1 appears to be dispensable for apoptosis. NUC-1 likely acts in a different DNA degradation pathway because cps-6, nuc-1 double mutants have higher numbers of TUNEL-positive cells than those of either mutant alone, as reported by Parrish et al. (2001). Recently, WAH-1, a C. elegans homolog of human apoptosis-inducing factor (AIF), was found to associate with and cooperate with cps-6 to promote DNA degradation in C. elegans, as reported in Wang et al. (2002). These results indicate that other unidentified proteins may be involved in the regulation of apoptotic DNA degradation in C. elegans. It is problematic that neither nuc-1 or cps-6 mutants nor wah-1 RNA-mediated interference animals display easily detectable phenotypes which would encourage additional genetic screens for mutants with similar cell death defects. Thus, there is a need to develop a more powerful and systematic method to identify molecular components that are involved in apoptotic DNA degradation. There is also a need to identify proteins that are involved in the execution of apoptosis and to gain knowledge on their mechanisms of action, because knowledge about these proteins may prove valuable for the diagnosis as well as treatment of diseases.